The last decade has seen an explosive growth in the knowledge of molecular biology and commercialization of that knowledge. Great success has been had in the cloning and expression of genes encoding proteins that were previously available in very small amounts, such as human growth hormone, tissue plasminogen activator and various lymphokines, to name just a few. Initially attempts were made to produce these proteins in bacterial or yeast expression systems. Many proteins may be preferably produced in cell culture. The reasons influencing one to use cell culture are: glycosylation of the desired protein, ease of purification of secreted products, and correct protein processing with correct folding and disulfide bond formation.
Once the gene encoding the desired protein is expressed in a mammalian cell line, its production must then be optimized. Optimization of protein yield in cell culture may be made by various means. Improvement may be obtained, for example by optimizing the physicochemical, nutritional, and hormonal environment of the cell.
Mammalian cells in vivo are in a carefully balanced homeostatic environment. The advantages of obtaining a completely defined medium for the growth of cells in vitro were recognized very early in the history of cell culture. (Lewis, M. R. and Lewis, W. H., Anat. Rec. 5:277 [1911]). Defined medium typically refers to the specific nutritional and hormonal chemicals comprising the medium required for survival or growth. Most cell types have stringent requirements as to the optimal range of physical parameters for growth and performance. Physicochemical parameters which may be controlled in different cell culture systems, for example, are: temperature, pH, pO.sub.2, and osmolarity. The nutritional requirements of cells are usually provided in standard media formulations developed to provide an optimal environment. Nutrients can be divided into several categories: amino acids and their derivatives, fatty acids, complex lipids, carbohydrates, sugars, vitamins and nucleic acid derivatives. Not only the absolute requirements, but the relative concentrations of nutrients must be optimized for each individual cell type.
Most cell types will not grow and/or secrete proteins optimally in medium consisting only of nutrients, even when the nutritional components are optimized. It is for this reason that serum has remained an essential medium component for the growth of cells in culture. Various experiments led to the hypothesis that the role of serum in cell culture was to provide a complex of hormones that were growth-stimulatory for a given cell type. (Sato, G. H. et al., in Biochemical Action of Hormones, Vol.III [G. Litwak, ed.] Academic Press, N.Y., page 391). A pituitary cell line was grown in serum-free medium supplemented with hormones, growth factors, and transferrin. (Hayashi, I. and Sato, G., Nature [Lond] 159:132 [1976]). Subsequently, hormone-supplemented serum-free conditions were developed for the growth of several cell lines originating from different tissues (Mather, J. and Sato, G., Exp. Cell Res. 120:191 [1979]; Barnes, D. and Sato, G., Cell 22:69 [1981]). These studies led to several conclusions concerning the growth of cells in serum-free medium. Serum can be replaced by a mixture of hormones, growth factors, and transport proteins. The required supplements (containing the hormones, growth factors and transport proteins) to serum-free medium may differ for different cell types. The supplements, traditionally, have been provided as part of complex biological mixtures such as serum or organ extracts. The "hormonal" milieu may be optimized to reduce or eliminate the need for undefined growth factors, remove inhibitory factors, or provide critical hormones at desirable levels.
Cells frequently require one or more hormones from each of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones. Most cell types require insulin to survive in serum-free media. (Sato, G. H. et al. in Growth of Cells in Hormonally Defined Media, [Cold Spring Harbor Press, N.Y., 1982]). Certain mutant cell lines have been reported which are insulin-independent. (Mendiaz, E. et al., In Vitro Cell. & Dev. Biol. 22 [2]:66 [1986]; Serrero, G., In Vitro Cell. & Biol. 21[9]:537 [1985]). In addition to the hormones, cells may require transport proteins such as transferrin (plasma iron transport protein), ceruloplasmin (a copper transport protein), and high density lipoprotein (a lipid carrier) to be added to cell media. The set of optimal hormones or transport proteins will vary for each cell type. Most of these hormones or transport proteins have been added exogenously or, in a rare case, a mutant cell line has been found which does not require a particular factor.
Recently, cellular proliferation has been studied to elaborate the events necessary to lead from quiescent growth arrest to the cellular commitment to proliferate. Various factors have been found to be involved in that transformation. These transformed cells have been found to produce peptide growth factors in culture. (Kaplan, P. L. et al., PNAS 79:485-489 [1982]). The secretion from a cell of a factor to which that same cell can respond has been referred to as an "autocrine" system. Numerous factors have been described as autocrine: bombesin, interleukin-2 (Duprez, V. et al. PNAS 82:6932 [1985]); insulin, (Serrero, G. In Vitro Cellular & Dev. Biol. 21 [9]:537 [1985]); transforming growth factor alpha (TGF-.alpha.), platelet-derived growth factor (PDGF); transforming growth factor-beta (TGF-.beta.), (Sporn, M. B. & Roberts, A. B., Nature 313:745 [1985]); sarcoma growth factor (SGF), (Anzano, M. A. et al., PNAS 80:6264 [1983]); and, hemopoietic growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF), (Lang, R. A. et al., Cell 43:531 [1985]).
It is an object of the present invention to provide a defined medium for particular recombinant host cells. Another object of this invention is to eliminate problems associated with the supply of necessary polypeptide factors for the maintenance and growth of recombinant host cells. For example, certain polypeptide factors, such as insulin, are unstable in some culture conditions. It is an object of the invention to provide a local environment for the host cell that is optimal for growth or survival. More particularly, it is an object of the invention to eliminate the need for preliminary testing, for example of purity, of polypeptide factors necessary for the host cells in cell culture. Yet another object of this invention is to lower the risk of contamination of a cell culture by eliminating the need of adding exogenous factors. Another object is to produce a more robust host cell line by providing autocrine production of polypeptide factors necessary for the survival and growth of recombinant host cells in culture. A further object is to produce recombinant host cells that are less sensitive to medium conditions. Still another object is to provide a localized environment for cell growth or survival. Yet another object is to improve the efficiency of cell culture through autocrine production of necessary polypeptide factors. And yet another advantage is to the lower the cost of the defined medium.